Optical-Isomer-Separating Agent

ABSTRACT

According to the present invention, there is provided an enantiomeric isomer separating agent, which includes a polysaccharide derivative supported by particles of a support by chemical bonding, and has a concentration of eluted component (in terms of mass proportion) as determined through a liquid passing test under the following conditions of 20 ppm or lower: (liquid passing test) (1) the enantiomeric isomer separating agent is packed into a column having a diameter of 1 cm and a length of 25 cm by a slurry method, (2) an organic solvent in which the polysaccharide derivative is soluble is used as a solvent, and passing of this solvent whose temperature is set at 40° C. is initiated at a flow rate of 8 ml/min, (3) collection of the solvent passed is initiated 1 hour after the initiation of the liquid passing, the solvent is collected for 1 hour (480 ml), and a mass (M 1 ) of the solvent is measured, and (4) the liquid collected is condensed and dried, a mass (M 2 ) of a residue is measured, and the concentration of eluted component is determined from an expression M 2 /M 1 .

TECHNICAL FIELD

The present invention relates to an enantiomeric isomer separating agentand a method of producing the agent.

BACKGROUND ARTS

A separating agent obtained by carrying a polysaccharide derivative onsilica gel has been generally used as, for example, an enantiomericresolution agent for high performance liquid chromatography. The mannersin each of which silica gel supports the polysaccharide derivative areclassified into the case where silica gel is caused to support thepolysaccharide derivative physically (physical support type separatingagent) and the case where silica gel is caused to support thepolysaccharide derivative by chemical bonding (chemical support typeseparating agent). See JP-B-2751004 or JP-A-8-5623.

The chemical support type separating agent has a smaller amount ofeluted component originating from the polysaccharide derivative thanthat of the physical support type separating agent even when an organicsolvent in which the polysaccharide derivative is soluble is used.However, an unreacted polysaccharide derivative is present even in thechemical support type separating agent, so an eluent contains elutedcomponent originating from the polysaccharide derivative.

In the case where eluted component originating from, for example, anunreacted polysaccharide derivative is present in an eluent as describedabove, the following problem arises depending on whether the amount ofeluted component is large or small when a separating agent containingsuch eluted component is used as a separating agent for high performanceliquid chromatography: the stability of the baseline of a chromatogramdeteriorates, or the purity of a fractionated product cannot besufficiently increased.

Investigations have already been conducted on a reduction in amount ofeluted component in the physical support type separating agent. JP-A7-260762 discloses a coating type separating agent having a small amountof eluted component.

DISCLOSURE OF THE INVENTION

A low-molecular-weight polysaccharide derivative is eluted in suchphysical support type separating agent. This is because the molecularweight distribution of a polysaccharide to be used varies.

On the other hand, not only such low-molecular-weight polysaccharidederivative as described above but also a low-molecular-weight orhigh-molecular-weight polysaccharide derivative that has not beenchemically bonded to a support, and, furthermore, an impurityoriginating from a compound having a functional group capable ofreacting with a hydroxyl group of a polysaccharide derivative are elutedin a chemical support type separating agent. As described above, elutedcomponent from a chemical support type polysaccharide derivative isdifferent from that of a physical support type polysaccharidederivative.

When a conventional chemical support type separating agent is used for aseparating agent for high performance liquid chromatography, asdescribed above, it still needs to be improved in terms of prevention ofthe deterioration of the stability of the baseline of a chromatogram,the stabilization of the quality of a fractionated product, and animprovement in purity of the fractionated product.

The present invention provides an enantiomeric isomer separating agentimproved in the separating performance when it is used for a separatingagent of high performance liquid chromatography and a method ofproducing the agent.

The present invention provides an enantiomeric isomer separating agent,which includes a polysaccharide derivative supported on particles of asupport by chemical bonding, and has a concentration of eluted component(in terms of mass proportion) as determined through a liquid passingtest under the following conditions of 20 ppm or lower:

(Liquid Passing Test)

(1) the enantiomeric isomer separating agent is packed into a columnhaving a diameter of 1 cm and a length of 25 cm by a slurry method,

(2) an organic solvent in which the polysaccharide derivative is solubleis used as a solvent, and passing of the organic solvent whosetemperature is set at 40° C. is initiated at a flow rate of 8 ml/min,

(3) collection of the organic solvent passed is initiated 1 hour afterthe initiation of the liquid passing, the organic solvent is collectedfor 1 hour (480 ml), and a mass (M₁) of the organic solvent is measured,and

(4) the liquid collected is condensed and dried, a mass (M₂) of aresidue is measured, and the concentration of eluted component isdetermined from an expression M₂/M₁.

According to the present invention, there is provided a method ofproducing the above-mentioned enantiomeric isomer separating agent,including the steps of: washing an unwashed enantiomeric isomerseparating agent with an organic solvent in which the polysaccharidederivative is soluble once or multiple times; and drying the washedenantiomeric isomer separating agent.

Further, according to the present invention, there is provided a methodof separating enantiomeric isomers including bringing the enantiomericisomers into contact with the above-mentioned enantiomeric isomerseparating agent, or use of the above enantiomeric isomer separatingagent for separating enantiomeric isomers.

DETAILED DESCRIPTION OF THE INVENTION

It should be noted that the term “unwashed enantiomeric isomerseparating agent” refers to an enantiomeric isomer separating agent thatdoes not satisfy the following requirement: the concentration of elutedcomponent (in terms of mass proportion) upon performance of a liquidpassing test under the above-mentioned conditions is 20 ppm or lower.

An enantiomeric isomer separating agent of the present invention hasgood separating performance, and can increase the purity of afractionated enantiomeric isomer. The agent is suitable for highperformance liquid chromatography.

(Enantiomeric Isomer Separating Agent)

The enantiomeric isomer separating agent of the present invention has aconcentration of eluted component (in terms of mass proportion) asdetermined through the following liquid passing test of 20 ppm or lower,preferably 15 ppm or lower, or more preferably 10 ppm or lower.

(Liquid Passing Test)

(1) the enantiomeric isomer separating agent is packed into a columnhaving a diameter of 1 cm and a length of 25 cm by a slurry method,

(2) an organic solvent in which the polysaccharide derivative is solubleis used as a solvent, and passing of the organic solvent whosetemperature is set at 40° C. is initiated at a flow rate of 8 ml/min,

(3) collection of the organic solvent passed is initiated 1 hour afterthe initiation of the liquid passing, the organic solvent is collectedfor 1 hour (480 ml), and a mass (M₁) for each fraction of the organicsolvent is measured, and

(4) the liquid collected is condensed and dried, a mass (M₂) of aresidue is measured, and the concentration of eluted component isdetermined from an expression M₂/M₁.

(Method of Producing Enantiomeric Isomer Separating Agent)

First, a chemical support type separating agent is produced bychemically bonding a polysaccharide derivative to a support.

A polysaccharide derivative to be used in the present invention is wellknown, and can be produced by applying, for example, Examples 1 to 5 ofJP-A-2004-3935, a method described in Synthesis Example 1 ofJP-A-7-260762, or a method described in Production Method 3 of paragraph43 of JP-B-2751004.

An enantiomeric isomer separating agent (unwashed enantiomeric isomerseparating agent) obtained by causing a support to support thepolysaccharide derivative to be used in the present invention is wellknown, and can be produced by applying, for example, a method describedin Example 1 of JP-B-2751004.

A porous organic support or a porous inorganic support can be used asthe support, and a porous inorganic support is preferable. A polymericsubstance composed of, for example, polystyrene, polyacrylamide, orpolyacrylate can be suitably used in a porous organic support. Silica,alumina, magnesia, glass, kaolin, titanium oxide, a silicate,hydroxyapatite, or the like can be suitably used in a porous inorganicsupport; silica gel is particularly preferable.

It should be noted that, when silica gel is used, silica gel isdesirably subjected to a surface treatment such as a silanizationtreatment (silanization treatment with an aminoalkylsilane) or a plasmatreatment in order that an influence of silanol remaining on the surfaceof silica gel may be eliminated, and the affinity of silica gel for anenantiomerically active polymer compound may be improved. However, noproblem arises even when the surface of silica gel is not treated atall.

A porous support, in particular, silica gel has a particle diameter ofpreferably 1 to 300 μm, more preferably 15 to 100 μm, or still morepreferably 20 to 50 μm, and has an average pore diameter of preferably200 to 8,000 Å, more preferably 200 to 4,000 Å, or still more preferably300 to 2,000 Å. It should be noted that the particle diameter of theporous support is substantially the particle diameter of the separatingagent.

The average pore diameter of the porous support preferably falls withinthe range because a solution of an enantiomerically active polymercompound is sufficiently infiltrated into the pores of the poroussupport, and the enantiomerically active polymer compound can easilyadhere to the inner wall of each of the pores in a uniform fashion.Further, the pressure loss of the separating agent can be maintained ata low level because the pores are prevented from clogging.

Examples of the polysaccharide conveying polysaccharide derivativeinclude any of a synthetic polysaccharide, a natural polysaccharide, anda natural products-modified polysaccharide as long as the polysaccharideor the derivative thereof is enantiomerically active. However, thepolysaccharide or the derivative thereof preferably has a highregularity in bonding manner.

Examples of the polysaccharide include: β-1,4-glucan (cellulose);α-1,4-glucan (amylose or amylopectin); α-1,6-glucan (dextran);β-1,6-glucan (pustulan); β-1,3-glucan (such as curdlan orschizophyllan); α-1,3-glucan; β-1,2-glucan (a Crown Gallpolysaccharide); β-1,4-galactan; β-1,4-mannan; α-1,6-mannan;β-1,2-fructan (inulin); β-2,6-fructan (levan); β-1,4-xylan; β-1,3-xylan;β-1,4-chitosan; α-1,4-N-acetylchitosan (chitin); pullulan; agarose; andalginic acid. Starch containing amylose is also included.

Of those, cellulose, amylose, β-1,4-xylan, β-1,4-chitosan, chitin,β-1,4-mannan, inulin, curdlan, and the like are preferred because theyeasily enables highly pure polysaccharides to be obtained. Cellulose andamylose are particularly preferred.

The number average degree of polymerization of the polysaccharide(average number of pyranose or furanose rings in one molecule) ispreferably 5 or more, or more preferably 10 or more, and has noparticular upper limit. However, the number average degree ofpolymerization is preferably 1,000 or less in terms of the easiness forhandling, and is more preferably 5 to 1,000, still more preferably 10 to1,000, or particularly preferably 10 to 500.

A product obtained by bonding a compound having a functional groupcapable of reacting with a hydroxyl group to some or all of the hydroxylgroups of the above-mentioned polysaccharide by, for example, esterbonding, urethane bonding or ether bonding can be used as thepolysaccharide derivative.

Examples of functional group-containing compounds that can react with ahydroxyl group include isocyanate derivatives, carboxylic acids, esters,acid halides, acid amide compounds, halides compounds, aldehydes,alcohols or other elimination group-containing compounds; and theiraliphatic, alicyclic, aromatic, and heteroaromatic compounds.

Examples of a particularly preferable polysaccharide derivative includea polysaccharide ester derivative and a polysaccharide carbamatederivative.

The term “eluted component” as used in the present invention refers to,for example, a reaction by-product such as an isocyanic acid derivativeor carboxylic acid derivative originating from a compound having afunctional group capable of reacting with a hydroxyl group, or apolysaccharide derivative that has not been chemically bonded to thesupport.

Next, the enantiomeric isomer separating agent obtained by chemicallybonding the polysaccharide derivative to the support (unwashedenantiomeric isomer separating agent) is washed with an organic solventin which the polysaccharide derivative is soluble. The washing can beperformed once or multiple times (for example, two to ten times).

A method of washing the unwashed separating agent may be any method withwhich the unwashed separating agent and the organic solvent can besufficiently brought into contact with each other, and a column mode, abatch mode, a method involving pouring the organic solvent into aresidue after filtration to wash the unwashed separating agent, aheating reflux method, or the like is applicable.

Examples of organic solvent used for washing can be any solvent selectedfrom tetrahydrofuran (THF), acetone, ethyl acetate, chloroform,methylene chloride, dimethyl acetoamide, dimethylformamide (DMF), andmethanol.

The total used amount of the organic solvent is preferably 5 to 100 ml,more preferably 10 to 70 ml, or still more preferably 20 to 40 ml withrespect to 1 g of the polysaccharide derivative in the unwashedseparating agent.

Whether the temperature of the organic solvent is lower than the boilingpoint of the solvent or not lower than the boiling point can be chosendepending on the washing method, and the temperature of the organicsolvent is preferably 50 to 150° C., more preferably 60 to 120° C., orstill more preferably 70 to 100° C.

The time needed for washing the unwashed separating agent may be anytime required to reduce the amount of eluted component to apredetermined amount or less, and is generally about 1 to 100 hours, orpreferably about 1 to 50 hours.

A filler after the washing can be recovered by, for example, filtration.The temperature at which the filler is filtrated is typically 10 to 150°C., or preferably 20 to 100° C.

Next, the separating agent after the washing is dried. A method ofdrying the separating agent is not particularly limited, and isappropriately selected in consideration of the time period for which theseparating agent is subjected to a drying treatment, and a cost for thetreatment. However, the separating agent is preferably subjected to adrying treatment under reduced pressure at room temperature to 100° C.for 2 to 24 hours.

An enantiomeric isomer separating agent obtained by the productionmethod of the present invention has a concentration of eluted componentin the above-mentioned liquid passing test of a predetermined value orlower. As a result, when the enantiomeric isomer separating agent of thepresent invention is used as a filler for high performance liquidchromatography to separate enantiomeric isomers, nearly no unreactedpolysaccharide derivative is eluted, so the baseline of a chromatogramat the time of fractionation is stabilized, and the purity of afractionated product can be increased. Accordingly, a high-qualityenantiomerically resoluble substance can be stably supplied.

EXAMPLES Example 1

(i) Synthesis of Amylose-Bonded Silica Gel

Under a nitrogen atmosphere, 100 g of silica gel the surface of whichhad been treated with a siliane treatment agent having an amino group atany one of its terminals and 20 g of amylose were dispersed in 380 ml ofDMSO at 70° C. A borane-pyridine complex and acetic acid were added tothe dispersion, and the whole was stirred for 20 hours. The stirredmixture was separated by filtration, and the residue was washed withmethanol, DMSO, and methanol in the stated order. After that, theresidue was dried in a vacuum, whereby amylose-bonded silica gel wasobtained.

(ii) Synthesis of enantiomeric isomer separating agent in which hydroxylgroup of amylose is turned into 3,5-dimethylphenylcarbamate

Under a nitrogen atmosphere, 100 g of amylose-bonded silica gel, 3 g of4-dimethylaminopyridine, 300 ml of DMF, and 45 g of3,5-dimethylphenylisocyanate were dispersed, and the dispersion wasstirred at 75° C. for 24 hours.

Amylose derivative-bonded silica gel thus obtained was washed with 2.4 Lof DMF at 75° C. (24 ml with respect to 1 g of an amylose derivative),whereby a target enantiomeric isomer separating agent was obtained.

(iii) A predetermined liquid passing test was performed by using theenantiomeric isomer separating agent of the present invention and ethylacetate as a solvent to determine the concentration of eluted component(ppm). As a result, the M₁ was 416.08 g, the M₂ was 1.2 mg, and theratio M₂/M₁ was 3 ppm.

Example 2

Silica gel the surface of which had been treated with a silane treatmentagent having an amino group at any one of its terminals was caused tosupport cellulose tris(3,5-dimethylphenylcarbamate) produced by a knownmethod (Example 1 of JP-B 2669554) by physical adsorption.

150 g of the resultant separating agent (ratio at which a polymer wassupported: 20 wt %) were suspended in 3 L of the mixed solvent of waterand acetonitrile, and the suspension was stirred. The suspension wasirradiated with UV for 220 minutes by a method described in Example 12of JP 11-510193 A so that crosslinking might take place. After that, theresultant solid was taken by filtration, repeatedly washed withtetrahydrofuran under heat and reflux 4 times, and dried in a vacuum,whereby a target enantiomeric isomer separating agent was obtained.

A predetermined liquid passing test was performed by using theenantiomeric isomer separating agent of the present invention andtetrahydrofuran as a solvent to determine the concentration of elutedcomponent (ppm). As a result, the M₁ was 416.18 g, the M₂ was 2.7 mg,and the ratio M₂/M₁ was 6.5 ppm.

Comparative Example 1

An unwashed chemical support type enantiomeric isomer separating agentwas obtained in the same manner as in Example 1. 100 g of the resultantseparating agent were washed by a general washing method. That is, theseparating agent was filtrated through a glass filter at roomtemperature, and was washed with 2.4 L of methanol.

A liquid passing test of the present invention was performed by usingthe resultant enantiomeric isomer separating agent and ethyl acetate asa solvent to determine the concentration of eluted component. The M₁ was416.15 g, the M₂ was 85 mg, and the ratio M₂/M₁ was 204 ppm.

Comparative Example 2

An unwashed chemical support type enantiomeric isomer separating agentwas obtained in the same manner as in Example 2. 100 g of the resultantseparating agent were washed by a general washing method. That is, theseparating agent was filtrated through a glass filter at roomtemperature, and was washed with 2.4 L of methanol.

A liquid passing test of the present invention was performed by usingthe resultant enantiomeric isomer separating agent and THF as a solventto determine the concentration of eluted component. The M₁ was 416.23 g,the M₂ was 203 mg, and the ratio M₂/M₁ was 488 ppm.

1. An enantiomeric isomer separating agent, which comprises apolysaccharide derivative supported by particles of a support bychemical bonding, and has a concentration of eluted component (in termsof mass proportion) as determined through a liquid passing test underthe following conditions of 20 ppm or lower: (liquid passing test) (1)the enantiomeric isomer separating agent is packed into a column havinga diameter of 1 cm and a length of 25 cm by a slurry method, (2) anorganic solvent in which the polysaccharide derivative is soluble isused as a solvent, and passing of the organic solvent whose temperatureis set at 40° C. is initiated at a flow rate of 8 ml/min, (3) collectionof the organic solvent passed is initiated 1 hour after the initiationof the liquid passing, the organic solvent is collected for 1 hour (480ml), and a mass (M₁) of the organic solvent is measured, and (4) theliquid collected is condensed and dried, a mass (M₂) of a residue ismeasured, and the concentration of eluted component is determined froman expression M₂/M₁.
 2. The enantiomeric isomer separating agentaccording to claim 1, wherein the polysaccharide derivative is acellulose derivative or an amylose derivative and the support is silicagel.
 3. A method of producing the enantiomeric isomer separating agentaccording to claim 1, comprising the steps of: washing an unwashedenantiomeric isomer separating agent with an organic solvent in whichthe polysaccharide derivative is soluble once or multiple times; anddrying the washed enantiomeric isomer separating agent.
 4. The method ofproducing the enantiomeric isomer separating agent according to claim 3,wherein the total of the used organic solvent is 5 to 100 ml withrespect to 1 g of the polysaccharide derivative in the enantiomericisomer separating agent.
 5. The method of producing the enantiomericisomer separating agent according to claim 3, wherein the organicsolvent used for the washing step is selected from the group consistingof tetrahydrofuran, acetone, ethyl acetate, chloroform, methylenechloride, dimethylacetamide and dimethylformamide.
 6. A method ofseparating enantiomeric isomers, comprising bringing enantiomericisomers into contact with the enantiomeric isomer separating agentaccording to claim
 1. 7. The method according to claim 6, wherein theseparation is performed by high performance liquid chromatography. 8.Use of the enantiomeric isomer separating agent according to claim 1 forseparating enantiomeric isomers.
 9. The use according to claim 8,wherein the enantiomeric isomer separating agent is used in highperformance liquid chromatography.